Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells

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Abstract

Reactive oxygen species are well known for induction of oxidative stress conditions through oxidation of vital biomarkers leading to cellular death via apoptosis and other process, thereby causing devastative effects on the host organs. This effect is believed to be linked with pathological alterations seen in several neurodegenerative disease conditions. Many phytochemical compounds proved to have robust antioxidant activities that deterred cells against cytotoxic stress environment, thus protect apoptotic cell death. In view of that we studied the potential of glucomoringin-isothiocyanate (GMG-ITC) or moringin to mitigate the process that lead to neurodegeneration in various ways. Neuroprotective effect of GMG-ITC was performed on retinoic acid (RA) induced differentiated neuroblastoma cells (SHSY5Y) via cell viability assay, flow cytometry analysis and fluorescence microscopy by means of acridine orange and propidium iodide double staining, to evaluate the anti-apoptotic activity and morphology conservation ability of the compound. Additionally, neurite surface integrity and ultrastructural analysis were carried out by means of scanning and transmission electron microscopy to assess the orientation of surface and internal features of the treated neuronal cells. GMG-ITC pre-treated neuron cells showed significant resistance to H2O2induced apoptotic cell death, revealing high level of protection by the compound. Increase of intracellular oxidative stress induced by H2O2 was mitigated by GMG-ITC. Thus, pre-treatment with the compound conferred significant protection to cytoskeleton and cytoplasmic inclusion coupled with conservation of surface morphological features and general integrity of neuronal cells. Therefore, the collective findings in the presence study indicated the potentials of GMG-ITC to protect the integrity of neuron cells against induced oxidative-stress related cytotoxic processes, the hallmark of neurodegenerative diseases.

Figures

  • Fig 1. Micrographs of neuronal cells differentiation by 10 μM all trans retinoic acid (ATRA). (a) Undifferentiated cells cultured in 10% complete growth media for seven (7) days and viewed under phase contrast, (b) Differentiated cells cultured in 3% heat-inactivated FBS complete growth media containing 10 μM ATRA for seven (7) days and viewed under phase contrast, and (d) expressed tuj-1 in both cytoplasm and neurites. SN = short neurites, EN = extended neurites, CYP = cytoplasm,. Magnification (x 20).
  • Fig 2. Cytotoxicity of GMG-ITC on differentiated neuronal cells at different concentrations (0.313 to 10) μg/ml. (A) display 24 h, (B) 48 h and (C) 72 h of treatment. Whereas (D) is a cytotoxic analysis result of H2O2 used in this study with IC50 = 300 μM. Values are presented in means ± SD of triplicate experiments and means with different letters varies significantly (p<0.05).
  • Fig 3. Concentration dependent viability of differentiated neuronal cells, pre-treated with GMG-ITC (0.313–10 μg/mL). (A) 24 h, (B) 48 h and (C) 72 h plus 4 h exposure to 300 μM H2O2. (D) is a means of 1.25 μg/ml GMG-ITC plus 4 h exposure to 300 μM H2O2. Values are presented in means ± SD of triplicate experiments and means with different letters varies significantly (p<0.05).
  • Fig 4. Acridine orange (AO, green) and propidium iodide (PI, red) double staining fluorescent micrographs of differentiated neuronal cells. (a) 4 h H2O2 treated cells, (b) 72 h myrosinase pre-treated plus 4 h H2O2 exposed cells, (c) 72 h 1.25 μg/ml GMG-ITC pre-treated plus 4 h H2O2 exposed cells, (d) untreated cells (normal control). The images were captured in multiple times and x20 magnification was used.
  • Fig 5. Annexin V-FITC assay of differentiated neuronal cells analysed by flow cytometry. Where (a) 4 h H2O2 treated cells, (b) 72 h myrosinase pre-treated plus 4 h H2O2 exposure cells, (c) untreated (normal control) cells, (d) GMG-ITC pre-treated for 24 h plus 4 h H2O2 exposure, (e) GMG-ITC pre-treated for 48 h plus 4 h H2O2 exposure and (f) GMG-ITC pre-treated for 72 h plus 4 h H2O2 exposure. Whereas (g) represent distribution of cells at death. Values are presented in means ± SD of triplicate experiments and means of viable cells with different letters varies significantly (p<0.05).
  • Fig 6. Surface morphological analysis of differentiated neuronal cells by scanning electron microscopy. (a) 4 h H2O2 treated cells, (b) 72 h myrosinase pre-treated plus 4 h H2O2 exposure cells, (c) 72 h GMG-ITC pre-treated plus 4 h H2O2 exposure cells, (d) untreated (normal control) cells. AB = apoptotic body, IDVC = intact differentiated viable cells, FN = folded neurites, MB = membrane blabbing, NDAC = neurite disrupted apoptotic cells. Magnification (x 5000).
  • Fig 7. Ultrastructural analysis of differentiated neuronal cells by transmission electron microscopy. (a) 4 h H2O2 treated cells, (b) 72 h myrosinase pre-treated plus 4 h H2O2 exposure cells, (c) 72 h GMG-ITC pre-treated plus 4 h H2O2 exposure cells, (d) untreated (normal control) cells. CM = chromatin margination, IN = intact nucleus, LD = lipid droplet, NC = nuclei convolution. Magnification (x 3000).

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CITATION STYLE

APA

Jaafaru, M. S., Nordin, N., Shaari, K., Rosli, R., & Abdull Razis, A. F. (2018). Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells. PLoS ONE, 13(5). https://doi.org/10.1371/journal.pone.0196403

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